CN110367542B - Probiotic microcapsule slowly released in intestinal tract and preparation method thereof - Google Patents
Probiotic microcapsule slowly released in intestinal tract and preparation method thereof Download PDFInfo
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- CN110367542B CN110367542B CN201910615451.9A CN201910615451A CN110367542B CN 110367542 B CN110367542 B CN 110367542B CN 201910615451 A CN201910615451 A CN 201910615451A CN 110367542 B CN110367542 B CN 110367542B
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- microcapsule
- probiotics
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- lactobacillus plantarum
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- 239000003094 microcapsule Substances 0.000 title claims abstract description 70
- 239000006041 probiotic Substances 0.000 title claims abstract description 53
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 53
- 230000000529 probiotic effect Effects 0.000 title claims abstract description 20
- 210000001035 gastrointestinal tract Anatomy 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims description 19
- 239000000463 material Substances 0.000 claims abstract description 39
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 24
- 239000005017 polysaccharide Substances 0.000 claims abstract description 24
- 150000004676 glycans Chemical class 0.000 claims abstract description 23
- 239000011162 core material Substances 0.000 claims abstract description 13
- 239000002994 raw material Substances 0.000 claims abstract description 3
- 240000006024 Lactobacillus plantarum Species 0.000 claims description 25
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims description 25
- 229940072205 lactobacillus plantarum Drugs 0.000 claims description 25
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims description 22
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 20
- 235000010413 sodium alginate Nutrition 0.000 claims description 20
- 239000000661 sodium alginate Substances 0.000 claims description 20
- 229940005550 sodium alginate Drugs 0.000 claims description 20
- 238000002156 mixing Methods 0.000 claims description 17
- 238000001914 filtration Methods 0.000 claims description 16
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- 238000006243 chemical reaction Methods 0.000 claims description 10
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- 239000001963 growth medium Substances 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 9
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- 239000002253 acid Substances 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
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- 238000009472 formulation Methods 0.000 description 2
- 210000002429 large intestine Anatomy 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 241000132012 Atractylodes Species 0.000 description 1
- 241000186012 Bifidobacterium breve Species 0.000 description 1
- 241001608472 Bifidobacterium longum Species 0.000 description 1
- 241001313855 Bletilla Species 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 229920002907 Guar gum Polymers 0.000 description 1
- 244000199885 Lactobacillus bulgaricus Species 0.000 description 1
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 description 1
- 241000186673 Lactobacillus delbrueckii Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
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- 244000057717 Streptococcus lactis Species 0.000 description 1
- 235000014897 Streptococcus lactis Nutrition 0.000 description 1
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- 102000004142 Trypsin Human genes 0.000 description 1
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- 239000001785 acacia senegal l. willd gum Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
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- 239000006185 dispersion Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 210000004211 gastric acid Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 235000010417 guar gum Nutrition 0.000 description 1
- 239000000665 guar gum Substances 0.000 description 1
- 229960002154 guar gum Drugs 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000003871 intestinal function Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 229940004208 lactobacillus bulgaricus Drugs 0.000 description 1
- BCZFSDNVXODRAJ-JTTNIQEDSA-N lycopodine Chemical compound C1CCN2CCC[C@@H]3[C@H]4C[C@@H](C)C[C@]32[C@H]1C(=O)C4 BCZFSDNVXODRAJ-JTTNIQEDSA-N 0.000 description 1
- BCZFSDNVXODRAJ-ZHMBSYLPSA-N lycopodine Natural products C1CCN2CCC[C@@H]3[C@H]4C[C@H](C)C[C@]32[C@H]1C(=O)C4 BCZFSDNVXODRAJ-ZHMBSYLPSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- -1 polysaccharide compound Chemical class 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
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- 238000013268 sustained release Methods 0.000 description 1
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- 239000012588 trypsin Substances 0.000 description 1
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- 235000010493 xanthan gum Nutrition 0.000 description 1
- 239000000230 xanthan gum Substances 0.000 description 1
- 229920001285 xanthan gum Polymers 0.000 description 1
- 229940082509 xanthan gum Drugs 0.000 description 1
- 239000005019 zein Substances 0.000 description 1
- 229940093612 zein Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Nutrition Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Dispersion Chemistry (AREA)
- Mycology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicinal Preparation (AREA)
- Manufacturing Of Micro-Capsules (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a probiotic microcapsule slowly released in intestinal tracts, which comprises a core material and a wall material, wherein the core material is probiotic, the wall material consists of a first wall material coated outside the core material and a second wall material coated outside the first wall material, the first wall material is sporopouenin, and the second wall material is high molecular polysaccharide. The invention can prevent the degradation and release of probiotics in stomach, thereby smoothly reaching intestinal tracts and slowly releasing and absorbing the probiotics, and has the advantages of good stability, high viable count, easily obtained raw materials, good biocompatibility and safety and the like, thereby having better application prospect.
Description
Technical Field
The invention relates to a probiotic microcapsule, in particular to a probiotic microcapsule which is slowly released in intestinal tracts, and a preparation method of the microcapsule.
Background
The probiotics have various health-care effects on human bodies, can improve intestinal functions, prevent cancers, enhance immune functions, regulate the level of cholesterol in the bodies and the like, but the exertion of a plurality of functions of the probiotics has great relation with the number of live bacteria and the living quality of the probiotics in the large intestine, and most probiotics preparations have two obvious problems: (1) after passing through digestive juice such as gastric acid, bile and the like, the activity of the probiotics is reduced; (2) most of the probiotics are released in the stomach and small intestine, and the amount of probiotics actually delivered to the large intestine is small.
In order to overcome the defects, the microcapsule technology is commonly adopted to improve the activity of probiotics, and in recent decades, extensive research is carried out on a preparation method of the microcapsule and abundant experience is obtained, however, how to rapidly and inexpensively manufacture the microcapsule with consistent size, porosity and structure, especially the microcapsule for oral probiotics still has many problems. The traditional single-layer embedding has low yield and poor embedding effect, can not play a good protection role on embedded active substances, and the microcapsule embedding technology with three or more layers improves the stability of thalli, but has complex process, is time-consuming and can not reasonably release bioactive components in intestinal tracts.
CN106617093A discloses an acid-resistant and stable probiotic microcapsule and a preparation method thereof, which is obtained by adding probiotic bacterial sludge into a mixed solution containing a natural polymer material and a freeze-drying protective agent, forming microdroplets and solidifying. The prepared probiotic microcapsule preparation has good viable count performance, good stability and acid resistance.
CN108618151A discloses a probiotic microcapsule for maintaining strain activity and a preparation method thereof, wherein a composite wall material structure consisting of a first wall material and a second wall material is adopted, the first wall material is a protein plant polysaccharide composite gel prepared from casein, a malt extract and xylo-oligosaccharide, damage to a core material by light, heat, oxygen, metal ions and the like can be well prevented, and a positive promotion effect on the stability of an embedding substance is achieved; the second wall material is a plant polysaccharide compound prepared from tremella polysaccharide, bletilla polysaccharide, white atractylodes rhizome polysaccharide, guar gum and pectin, and can greatly reduce the influence of a vacuum freeze-drying processing technology on the biological activity of the core material probiotic strain; meanwhile, when the first wall material is wrapped with the second wall material, the first wall material containing a large amount of protein can increase the film forming property and the embedding effect of the second wall material, so that the performance of the composite wall material is integrally improved, and the composite wall material plays roles in better protecting the biological activity of the core material and obviously improving the stability of the core material.
None of the above methods report the release effect of microcapsules in the intestinal environment.
In nature, sporopollenin is the main component of the outer wall of pollen and is responsible for protecting genetic materials under different environmental conditions and ensuring the reproductive capacity of plants. Sporopouenin obtained from natural plant spore has the advantages of uniform structure, stable mechanical and chemical properties, monodispersity, large porosity, large inner cavity, good biocompatibility, natural degradation in human body and livestock body, no generation of secondary harmful substances, and the like, and is a natural drug delivery system.
CN108434119A discloses a method for preparing a protein oral microcapsule preparation, which comprises three steps of sporopouenin preparation, medicine loading and sporopouenin outer wall encapsulation. The protein oral microcapsule is prepared by taking sunflower sporopollenin as a capsule wall material, and then the zein is adopted to block pores on the sporonin outer wall, so that the degradation and release of therapeutic protein in the stomach are reduced, and the therapeutic protein can smoothly pass through the stomach and be absorbed by intestinal tracts.
CN108836950A discloses another method for preparing oral protein microcapsules, which comprises dissolving lycopodium sporopollen and protein in water for embedding, so that the protein is embedded in the cavity structure of sporopollen, then dissolving sporopollen loaded with drugs in a high molecular polysaccharide solution, plugging the cavity structure of sporopollen with the high molecular polysaccharide, and then adding a cross-linking agent to make the high molecular polysaccharide perform cross-linking reaction and solidify, thereby reducing the degradation and release of therapeutic protein in stomach, and making it pass through stomach smoothly and be absorbed.
However, since the probiotics are living microorganisms and have very special properties, the sporopouenin cannot be directly used for loading the probiotics, and in addition, because the stability of the probiotics is poor, the microcapsules with high viable count are difficult to obtain by adopting the method.
Disclosure of Invention
The present invention has been made in view of the above-mentioned problems occurring in the prior art, and an object of the present invention is to provide a probiotic microcapsule having sporopouenin as a wall material, which is prevented from being degraded and released in the stomach, smoothly reaches the intestinal tract, and is slowly released and absorbed.
The probiotic microcapsule slowly released in intestinal tract comprises a core material and a wall material, wherein the core material is probiotic, the wall material consists of a first wall material coated outside the core material and a second wall material coated outside the first wall material, the first wall material is sporopouenin, and the second wall material is high molecular polysaccharide.
The probiotics comprise lactobacillus plantarum, lactobacillus bulgaricus, lactobacillus delbrueckii, bifidobacterium longum, bifidobacterium breve, streptococcus thermophilus, lactococcus lactis and the like. The high molecular polysaccharide comprises xanthan gum, Arabic gum, gelatin, sodium alginate, chitosan, pachyman, etc., wherein the pachyman is insoluble in water, has low biological activity, and can be converted into carboxyl pachyman through carboxymethylation, so that the solubility can be increased, and the biological activity can be improved.
The invention further provides a preparation method of the probiotic microcapsule, which comprises the following steps:
1) mixing sporopollen with a probiotic culture medium, sterilizing, cooling to normal temperature, inoculating probiotics, performing proliferation culture, and filtering to obtain primary microcapsules;
2) adding the primary microcapsule into the high molecular polysaccharide solution, uniformly mixing, adding a cross-linking agent to enable the high molecular polysaccharide to generate cross-linking reaction and solidify, and freeze-drying the precipitate to obtain the microcapsule.
The sporopouenin is lycopodine or Helianthus annuus sporopouenin, and can also be sporopouenin of other plants, preferably Helianthus annuus sporopouenin.
When the probiotics is lactobacillus plantarum, the culture medium is MRS liquid culture medium, the temperature of proliferation culture is 30 ℃, the time is 14h, the weight volume ratio of sporopollen to MRS liquid culture medium is 100 mg: 50-150mL, preferably 100 mg: 50 mL.
The weight volume ratio of the sporopouenin to the high molecular polysaccharide solution is 100 mg: 10-30ml, preferably 100 mg: 20 ml.
Preferably, the high molecular polysaccharide consists of sodium alginate and carboxymethyl pachyman, and the weight ratio of the sodium alginate to the carboxymethyl pachyman is 2-5: 1-3, preferably 3.3: 2.
preferably, the carboxymethyl pachyman has a substitution degree of 0.9, a molecular weight of 120000Da and a viscosity of 1.03 mPas.
According to one embodiment of the present invention, the optimal preparation method of lactobacillus plantarum microcapsules is as follows:
(1) embedding of Lactobacillus plantarum
Mixing 100mg Helianthus annuus sporopollen with 50mL MRS liquid culture medium, sterilizing at 121 deg.C for 15min, cooling to room temperature, inoculating Lactobacillus plantarum strain, performing proliferation culture at 30 deg.C for 14h, and filtering to obtain primary microcapsule.
(2) Preparation of double-layer microcapsules
Dissolving sodium alginate and carboxymethyl pachyman (with the substitution degree of 0.9, the molecular weight of 120000Da and the viscosity of 1.03mPa & s) in water to prepare a mixed solution with the weight content of 3.3 percent of sodium alginate and the weight content of 2 percent of carboxymethyl pachyman, then adding the primary microcapsule obtained in the step (1) into 20mL of the mixed solution, uniformly mixing, dripping 0.05mol/L of calcium chloride solution to enable the high polymer material to generate a crosslinking reaction and solidify, standing for 10min, filtering, washing the precipitate with normal saline, and freeze-drying to obtain the lactobacillus plantarum double-layer microcapsule.
Compared with the prior art, the invention has the following advantages:
firstly, the sporopouenin is used as a wall material to embed the probiotics, compared with other materials, the wall material is derived from natural plants, is easy to obtain raw materials, can be naturally degraded in human bodies and livestock bodies, does not generate secondary harmful substances, has better biocompatibility and safety, has the advantages of stable property, uniform dispersion, large porosity and inner cavity and the like, can slowly release the probiotics, is beneficial to improving the stability of the probiotics, and blocks the influence of external adverse environmental factors on the probiotics.
Secondly, the invention overcomes the defects of the sporopouenin embedding process in the prior art, adopts a co-culture method completely different from the prior art, and compared with methods such as vacuum adsorption and the like, the method not only realizes the loading of probiotics, but also has higher embedding efficiency and can load more viable bacteria.
And the second wall material is used for wrapping, so that the stability of the probiotics is further improved, the inactivation of the probiotics in the stomach is prevented, and the viable bacteria can smoothly reach the intestinal tract and be released.
Finally, after the probiotic microcapsule is stored for 60 days at 4 ℃, the viable count can be kept at 10 8 More than CFU/g, thereby having higher storage stability.
In a word, the sporopollen microcapsule provided by the invention not only has all the advantages of sporopollen, but also overcomes the adverse factors brought by probiotics, realizes the preparation of the probiotics microcapsule and the slow release of the probiotics microcapsule in intestinal tracts, and has good application prospects in human and animal health care and disease treatment.
Detailed Description
The invention will be further illustrated below by taking Lactobacillus plantarum as an example. Helianthus annuus sporopollenin and lycopodium sporonin used in the following examples are prior art and disclosed in patent documents CN108434119A and CN108836950A, respectively.
Example 1
(1) Embedding of Lactobacillus plantarum
Adopting a co-culture method: mixing 1000mg Helianthus annuus sporopollen with 500mL MRS liquid culture medium, sterilizing at 121 deg.C for 15min, cooling to room temperature, inoculating Lactobacillus plantarum strain, performing proliferation culture at 30 deg.C for 14h, and filtering to obtain primary microcapsule.
And placing the primary microcapsules in simulated intestinal fluid, incubating for 3.5 hours in a constant-temperature shaking table at 37 ℃, and then diluting, separating and counting the lactobacillus plantarum in the simulated intestinal fluid. The preparation method of the simulated intestinal juice comprises the following steps: mixing 6.8g/L KH 2 PO 4 The pH was adjusted to 7.5. + -. 0.1 with NaOH, sterilized and 10g/L trypsin was added. The number of the living bacteria loaded in the primary microcapsule is 2.34 multiplied by 10 through detection and calculation 10 CFU/g。
(2) Preparation of double-layer microcapsules
Dissolving sodium alginate and carboxymethyl pachyman (with the substitution degree of 0.9, the molecular weight of 120000Da and the viscosity of 1.03mPa & s) in water to prepare a mixed solution with the weight content of 3.3 percent of sodium alginate and the weight content of 2 percent of carboxymethyl pachyman, then adding the primary microcapsule obtained in the step (1) into 200mL of the mixed solution, uniformly mixing, dripping 0.05mol/L of calcium chloride solution to enable the high polymer material to generate a crosslinking reaction and solidify, standing for 10min, filtering, washing the precipitate with normal saline, and freeze-drying to obtain the lactobacillus plantarum double-layer microcapsule.
Example 2
(1) The primary microcapsules were prepared using lycopodium and the other methods and conditions were the same as in step (1) of example 1. Through inspectionThe number of the loaded viable bacteria is 7.28 multiplied by 10 6 CFU/g. The entrapment capacity of the Helianthus annuus sporopollen to the Lactobacillus plantarum is obviously larger than that of the lycopodium sporopollen, which is probably because of the unique structure of the Helianthus annuus sporopollen, not only micro-pores but also nano-pores exist, and probiotics enter the cavity of the microcapsule more.
Example 3
(1) Preparing primary microcapsules by a vacuum adsorption method: taking 500mL of cultured lactobacillus plantarum bacterial liquid, centrifuging for 10min under 3000r/min to obtain bacterial sludge, dissolving the bacterial sludge and 1000mg of Helianthus annuus sporopollen in 30mL of physiological saline water, carrying out vacuum treatment for 3h under the conditions of 2mbar and 25 ℃, filtering, washing and drying.
The detection shows that the number of the loaded viable bacteria is 1.32 multiplied by 10 2 CFU/g, essentially without embedding.
Example 4
(1) Mixing 1000mg Helianthus annuus sporopollen with 1500mL MRS liquid culture medium, sterilizing at 121 deg.C for 15min, cooling to room temperature, inoculating Lactobacillus plantarum strain, and performing proliferation culture at 37 deg.C for 7 h. Filtering and drying to obtain the primary microcapsule.
The detection shows that the number of the loaded viable bacteria is 1.05 multiplied by 10 8 CFU/g。
Example 5
(1) Mixing 1000mg Helianthus annuus sporopollen with 1000mL MRS liquid culture medium, sterilizing at 121 deg.C for 15min, cooling to room temperature, inoculating Lactobacillus plantarum strain, and performing proliferation culture at 28 deg.C for 21 h. Filtering and drying to obtain the primary microcapsule.
The detection shows that the number of the loaded viable bacteria is 1.71 multiplied by 10 9 CFU/g。
Example 6
(1) The method and conditions were the same as in step (1) of example 1.
(2) Dissolving sodium alginate and carboxymethyl pachyman (with the substitution degree of 0.9, the molecular weight of 120000Da and the viscosity of 1.03mPa & s) in water to prepare a mixed solution with the weight content of sodium alginate of 2.5% and the weight content of carboxymethyl pachyman of 3%, then adding the primary microcapsule obtained in the step (1) into 100mL of the mixed solution, uniformly mixing, dripping 0.05mol/L of calcium chloride solution to enable the high polymer material to generate a crosslinking reaction and solidify, standing for 10min, filtering, washing the precipitate with normal saline, and freeze-drying to obtain the lactobacillus plantarum double-layer microcapsule.
Example 7
(1) The method and conditions were the same as in step (1) of example 1.
(2) Dissolving sodium alginate and carboxymethyl pachyman (with the substitution degree of 0.7, the molecular weight of 330000Da and the viscosity of 1.53mPa & s) in water to prepare a mixed solution with the weight content of sodium alginate of 3.75% and the weight content of carboxymethyl pachyman of 1.5%, then adding the primary microcapsule obtained in the step (1) into 300mL of the mixed solution, uniformly mixing, dripping 0.05mol/L of calcium chloride solution to enable the high polymer material to generate a crosslinking reaction and solidify, standing for 10min, filtering, washing the precipitate with physiological saline, and freeze-drying to obtain the lactobacillus plantarum double-layer microcapsule.
Example 8
(1) The method and conditions were the same as in step (1) of example 1.
(2) Dissolving sodium alginate and carboxymethyl pachyman (with the substitution degree of 0.7, the molecular weight of 330000Da and the viscosity of 1.53mPa & s) in water to prepare a mixed solution with the weight content of 4.2% of sodium alginate and 1% of carboxymethyl pachyman, then adding the primary microcapsule obtained in the step (1) into 150mL of the mixed solution, uniformly mixing, dripping 0.05mol/L of calcium chloride solution to enable a high polymer material to generate a crosslinking reaction and solidify, standing for 10min, filtering, washing a precipitate with normal saline, and freeze-drying to obtain the lactobacillus plantarum double-layer microcapsule.
Example 9
(1) The method and conditions were the same as in step (1) of example 1.
(2) Dissolving sodium alginate in water to prepare a sodium alginate solution with the weight content of 5%, then adding the primary microcapsule obtained in the step (1) into 200mL of the solution, uniformly mixing, dripping 0.05mol/L of calcium chloride solution to enable the high polymer material to generate a crosslinking reaction and solidify, standing for 10min, filtering, washing the precipitate with physiological saline, and freeze-drying to obtain the lactobacillus plantarum double-layer microcapsule.
Comparative example 1
(1) And taking 20mL of activated and cultured lactobacillus plantarum bacterial liquid. Centrifuging at 4 deg.C and 3000rpm for 10min, and removing supernatant to obtain bacterial sludge.
(2) Dissolving sodium alginate and carboxymethyl pachyman (with the substitution degree of 0.9, the molecular weight of 120000Da and the viscosity of 1.03mPa & s) in water to prepare a mixed solution with the weight content of sodium alginate of 3.3% and the weight content of carboxymethyl pachyman of 2%, then adding the bacterial sludge obtained in the step (1) into 200mL of the mixed solution, uniformly mixing, dripping 0.05mol/L of calcium chloride solution to enable the high polymer material to generate a crosslinking reaction and solidify, standing for 10min, filtering, washing the precipitate with normal saline, and freeze-drying to obtain the lactobacillus plantarum microcapsule.
Comparative example 2
Mixing 1000mg Helianthus annuus sporopollen with 500mL MRS liquid culture medium, sterilizing at 121 deg.C for 15min, cooling to room temperature, inoculating Lactobacillus plantarum strain, and performing proliferation culture at 30 deg.C for 14 h. Filtering and freeze-drying to obtain the primary microcapsule.
Examples of the experiments
1. Detection of viable count of probiotics in microcapsules
Counting by a viable bacteria counting method, detecting the viable bacteria number of the probiotic microcapsule preparation after freeze-drying, and the result is shown in table 1.
TABLE 1 comparison of viable count in lyophilized microcapsules
From the results, the double-layer microcapsule prepared by adopting the high molecular polysaccharide has higher viable count, and the comparative example 2 does not adopt the high molecular polysaccharide to wrap, so that the viable count after freeze-drying is greatly reduced. Wherein, the ratio of sodium alginate to carboxymethyl pachyman, the dosage of polysaccharide solution and the parameters of carboxymethyl pachyman all have certain influence on the number of viable bacteria in the preparation, and in example 9, because carboxymethyl pachyman is not added, the protection effect is poor, and the number of viable bacteria after freeze-drying is relatively low.
2. Release behavior of probiotic microcapsules under simulated digestion conditions
Taking 10g of microcapsules in 90mL of simulated gastric juice, treating the microcapsules in a constant-temperature air shaking table at 37 ℃ for 2 hours, filtering the microcapsules, dividing the microcapsules into 9 groups and 1g of the microcapsules, subpackaging the microcapsules in 9 bacterial bottles, adding 9mL of simulated intestinal juice in each bacterial bottle, placing the bacterial bottles in the constant-temperature air shaking table at 37 ℃, taking out one bacterial bottle at nine time points of 5min, 15min, 30min, 60min, 90min, 120min, 180min, 270min and 360min, counting the number of viable bacteria in the simulated intestinal juice by using a dilution separation counting method, and calculating the release rate by combining the data in the table 1. The formulation of the simulated gastric juice is that 9g/L NaCl solution is adjusted to pH 2.0 by hydrochloric acid, and pepsin of 3.2g/L is added after sterilization; the formulation of simulated intestinal fluid was the same as in example 1. The results are shown in table 2:
TABLE 2 simulation of cumulative Release Rate of Lactobacillus plantarum in intestinal juice
| Group of | Simulated intestinal juice 30min viable bacteria release rate (%) | Simulated cumulative Release Rate in intestinal fluid (%) |
| Example 1 | 24.2 | 92.8 |
| Example 6 | 31.7 | 85.9 |
| Example 7 | 28.9 | 87.4 |
| Example 8 | 35.3 | 89.2 |
| Example 9 | 41.2 | 75.6 |
| Comparative example 1 | 59.8 | 62.4 |
| Comparative example 2 | 11.7 | 13.5 |
From the above results, it can be seen that comparative example 2 has no protection of the second wall material, so that the probiotics are inactivated in a large amount after being digested by gastric juice, and the number of viable bacteria entering intestinal juice is small, so that the cumulative release rate is low, and the probiotics are released in the intestinal juice quickly and hardly have a sustained release effect. Comparative example 1 the release of viable bacteria in the intestine was rapid and the number of bacteria released in 30 minutes was close to the peak value, since no sporopouenin was used for encapsulation. The double-layer microcapsule prepared by adopting the macromolecular polysaccharide can ensure that most of probiotics are released in intestinal tracts, and can obviously reduce the release speed of the probiotics in the intestinal tracts.
Claims (1)
1. The probiotic microcapsule slowly released in intestinal tract comprises a core material and a wall material, wherein the core material is lactobacillus plantarum, and is characterized in that: the wall material consists of a first wall material coated outside the core material and a second wall material coated outside the first wall material, wherein the first wall material is sunflower sporopollen, and the second wall material is high molecular polysaccharide, and the preparation method comprises the following steps:
1) sunflower sporopollen and MRS liquid culture medium are mixed according to the weight volume ratio of 100 mg: mixing 50mL of the raw materials according to a proportion, sterilizing, cooling to the normal temperature, inoculating lactobacillus plantarum, performing proliferation culture at the temperature of 30 ℃ for 14 hours, and filtering to obtain primary microcapsules;
2) adding primary microcapsules into a high molecular polysaccharide solution, wherein the weight-volume ratio of the sunflower sporopollenin to the high molecular polysaccharide solution is 100 mg: 20ml, wherein the high molecular polysaccharide consists of sodium alginate and carboxymethyl pachyman, and the weight ratio of the sodium alginate to the carboxymethyl pachyman is 3.3: 2, the substitution degree of the carboxymethyl pachyman is 0.9, the molecular weight is 120000Da, the viscosity is 1.03mPa & s, the cross-linking agent is added after the uniform mixing, the high-molecular polysaccharide is subjected to cross-linking reaction and solidification, and the precipitate is freeze-dried to obtain the carboxymethyl pachyman.
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